The human lens must maintain transparency over many decades and, in order to do so, fiber cells must establish close packing and preserve protein solubility. Lens membrane proteins, such as transporters and adhesion molecules, play essential roles in lens development and maintenance of homeostasis. Aquaporin-0 (AQPO) and MP20, the most abundant lens membrane proteins, have reported roles in fiber cell adhesion, in water permeability, and in gap junctional organization and, as such, play important roles in the development and maintenance of lens transparency. The long-term goal of our research is to identify modifications to the lens membrane proteome during development and aging and to understand how lens membrane protein function is altered by modification. Our general hypothesis is that modifications to AQPO and MP20 alter protein function in specific lens regions during lens development and accumulate with age leading to cataracts. More specifically, we hypothesize that AQPO C-terminal modifications regulate calmodulin binding and alter a newly discovered interaction withlens specific cytoskeletal elements in specific regions of the lens. To test this hypothesis our goal is to use state-of-the-art proteomics and microscopy approaches to generate a molecular level understanding of how AQPO and MP20 are modified in distinct stages of fiber cell development. We will then determine how these modifications affect AQPO protein-protein interactions. We propose four aims: 1) To identify AQPO modifications in normal and cataractous human lenses with high spatial resolution corresponding to different stages of fiber cell differentiation and age, and 2) To determine the sites of interaction between AQPO and filensin/CP49, the effect of AQPO modification on this interaction, and the in vivo effects of loss of this interaction, 3) To determine the effects of AQPO modification on interactions with calmodulin, and 4) To identify MP20 modifications in normal and cataractous human lenses with high spatial resolution corresponding to different stages of fiber cell differentiation and age. The global approach proposed is expected to provide new detailed information on the structure and function of the most abundant integral membrane proteins in the lens and lead to an improved understanding of normal lens development, aging processes, and cataractogenesis.